1. Field of the Invention
This invention concerns a laser-based device for volatilizing quantitatable amounts of nonvolatile solid organic compounds. In a preferred embodiment, this laser volatilizing device is coupled to a second laser capable of ionizing the volatilized molecules to provide a laser-based ion source which generates reproducible bursts of ions from nonvolatile solid organic compounds with negligible fragmentation. The ions so generated can be used to accurately quantitate the organic compounds. In the present device, a pulse of energy from a first laser generates a cloud of volatilized molecules of the solid analyte off of a support selected to permit and facilitate rapid, localized heating. In the ion source a pulse of energy from a second laser reproducibly ionizes a portion of the molecules in the cloud. In preferred embodiments this ion source is used to generate ions which are resolved in a mass spectrometer apparatus. The use of this ion source permits femtomoles or less of nonvolatile molecules to be quantitated in mass spectrometers.
2. Background Information
Chemical analyses can be carried out to identify or "qualitate" materials in a sample. Analyses can also be carried out to not only identify materials in a sample but also measure the amounts of these materials. This latter type of analysis is referred to as a "quantitative" analysis or as "quantitation". To permit quantitation, an analytical technique must generate a result or signal which is reproducible in magnitude as well as in identity and which is related to the amount of analyte. The present invention provides a laser-based volatilizer and ion source which generates volatilized molecules and ions of nonvolatile solid organic compounds with such a level of precision and reproducibility that, for the first time, it is possible to base quantitative analytical methods upon the ions so generated. Because this invention involves both the field of lasers and the field of ion-based analytical methods such as mass spectrometry, in this Background section art related to laser-based analytical techniques will be presented first, followed by art related to ion-based analytical methods.
The development of laser-based analytical methods in recent years has been rapid. Recent general reviews of these advances include Hieftje, G.M., Travis, J.C., Lytle, F.E., eds., "Lasers in Chemical Analysis," The Humana Press: Clifton, NJ, 1981; Kliger, D.S., ed., "Ultrasensitive Laser Spectroscopy"; Academic Press: New York, 1983; Keller, R.A., ed., "Laser-Based Ultrasensitive Spectroscopy and Detection V," Proc. Soc. Phot-Opt. Instrum. Eng. 1983, 426; and Zare. R.N., Science 1984, 226, 298; Delgass, W.N., Cooks, R.G., Science 1987, 235, 54-5.
particularly dramatic has been the explosive growth in laser-assisted mass spectrometry. A very complete review of this area entitled "A Review of the Application to Solids of the Laser Ion Source in Mass Spectrometry" was published by Conzemius. R.J., and Capellen, J.M., in Int. J. Mass Spec. Ion Phys., 1980, 34, 197, while an equally thorough review of recent work may be found at Burlingame. A.L., Baillie, T.A., Derrick. P.J., Anal. Chem. 1986, 58(5), 165R.
Mass spectral analysis has become increasingly important as a method of identification of materials. A most severe limitation in the mass spectral analysis of thermally labile or highly polar compounds is that thermal evaporation of the sample is required. In most cases the energy needed for the evaporation step exceeds that for thermal degradation. Primarily for this reason, many biologically important substances have proven intractable in analysis by classical mass spectrometric methods. To address this problem, a number of less destructive techniques have been proposed to facilitate the introduction of these materials into mass spectrometry. These include condensed-phase ejection-ionization methods like the improved thermal desorption method (Rizzo. T.R., Park. Y.D., Peteanu. L.A., Levy, D.U., J. Chem. Phys. 1986, 84, 2534); field desorption (Beckey. H.D., "Principles of Field Ionization and Field Desorption Mass Spectrometry," Pergamon Press: New York, 1977); Plasma desorption (Benninghoven. A., ed., "Ion Formation from Organic Solids," Springer-Verlag: Berlin. 1983, part 2); sputtering of substances as secondary ions by bombardment with energetic primary ions (SIMS) (Benninghoven, A., Sichtermann, W.K., Anal. Chem. 1978, 50, 1180; Benninghoven, A., Colton, R.J., Simons, D.S., & Werner, H.W., eds., "Secondary Ion Mass Spectrometry SIMS V," Springer Series in Chemical Physics 44, Springer-Verlag: Berlin, 1986); sputtering of substances as secondary ions by bombardment with atoms (FAB) (Benninghoven. A., ed., "Ion Formation from Organic Solids," Springer-Verlag: Berlin, 1983, part 3); thermospray (Blakley, C.R., Vestal, M.L., Anal. Chem. 1983. 55, 750; Vestal, M.L., Fergusson. G.J., Anal. Chem. 1985, 57, 2373); and electrospray (Whitehouse, C.M., Dreyer, R.N., Yamashita, M., Fenn, J.B., Anal. Chem. 1985, 57, 675). The general concept of using a laser to directly generate ionic species from solids also has been suggested by a number of laboratories (see, for example, Posthumus. M.A., Kistemaker, P.G., Meuzelaar, H.L.C., Anal. Chem. 1978, 50, 985; Stoll, R., Rollgen, F.W., Org. Mass Spec. 1979, 14, 642; Antonov, V.S., Letokhov. V.S., Shibanov, A.N., Appl. Phys. 1981, 25, 71; Antonov. V.S., Letokhov, V.S., Matveyets. YU.A., Shibanov, A.N., Laser Chem. 1982, 1, 37; Tabet, J.-C., Cotter, R.J., Anal. Chem. 1984, 56. 1662; Egorov, S.E., Letokhov. V.S., Shibanov. A.N., Chem. Phys. 1984, 85, 349; Deviney, M.L., Gland, J.L., eds., "Catalyst Characterization Science." American Chemical Society: Washington. D.C., 1985, pp. 238-251; Karas. M., Bahr, U., Trends in Anal. Chem. 1986, 5, 90: Sherman M.G., Kingsley, J.R., Hemminger, J.C., Mclver, R.T., Jr., Anal. Chim. Acta 1985, 178, 79; Wilkins. C.L., Weil, D.A., Yang, C.L.C., Ijames, C.F., Anal. Chem. 1985. 57. 520; Brown, R.S., Wilkins, C.L., Anal. Chem. 1986, 58. 3196; Brown, R.S., Wilkins, C.L., J. Am. Chem. Soc. 1986. 108. 2447; Coates, M.L., Wilkins, C.L., Anal. Chem. 1987, 59, 197: Holm, R., Karas, M., Vogt, H., Anal. Chem. 1987, 59, 373).
All these techniques have in common that ions are created directly out of the condensed phase by the impact of the bombarding particle, i.e., the fast atoms (Benninghoven, ed., part 3), the ions (Benninghoven, Sichtermann; Benninghoven. Colton et al., eds.), or the laser light photons (Conzemius. R.J., Capellen. J.M., Int. J. Mass Spec. Ion Phys. 1980, 34, 197; Hercules. D.M., Day. R.J., Balasanmugam, K., Dant, T.A., Li, C.P., Anal. Chem. 1982, 54, 280A; Hercules, D.M., Pure & Appl. Chem. 1983, 55, 1869: as well as posthumus et al.; Stoll el al.; Anronov et al., Appl. Phys.; Antonov et al., Laser Chem.; Egorov et al.; Deviney et al., eds; Karas et al.; Sherman et al.; Wilkins et al.; Brown et al., Anal. Chem.; Brown et al., J. Am. Chem. Soc.; Coates et al.; Holm et al.). Since single-step processes all rely on a single impact to bring about desorption and ionization, there has been no ability to independently vary desorption or ionization conditions. This has led to problems with reproducibility from analysis to analysis on a given sample as well as with variation in efficiency from sample to sample. These problems interfere with the ability to carry out a quantitative analysis with this method.
One improvement in laser ion generation techniques has been to divide the process into two separate steps--a desorption step to generate gaseous particles (molecules or atoms) from the solid, followed by ionization of the gaseous particles in a second step with a separate second laser pulse. This is possible because most of the desorbed material is in the neutral rather than in the ionic state, often with an ion-to-neutral ratio in the range of 10.sup.-3 to 10.sup.-5. Descriptions of prior multistep systems can be found in Antonov, V.S., Letokhov, V.S., Matveyets, YU. A., Shibanov, A.N., Laser Chem. 1982, 1, 37; Antonov, V.S., Egorov, S.E., Letokhov, V.S., Shibanov, A.N., JETP Lett. 1983, 38, 217; Becker, C.H., Gillen, K.T., Anal. Chem. 1984, 56. 1671; Becker, C.H., Gillen, K.T., J. Opt. Soc. Am. B 1985, 2, 1438: Nogar, N.S., Estler, R.C., Miller, C.M., Anal. Chem. 1985, 57, 2441; and Tembreull, R., Lubman. D.M., Anal. Chem. 1986, 58, 1299; Frey, R., Weiss, G., Kaminski. H., Schlag, E.W., Z. Naturforsch. Teil A 1985, 40, 1349; Walter, K., Bosel, U., Schlag, E.W., Int. J. Mass Spectrom. Ion Pro. 1986, 71, 309; Grotemeyer, I., Bosel, U., Walter, K., Schlag, E.W., J. Am. Chem. Soc. 1986, 108. 4233.
The Antonov et al. system employs a high power CO.sub.2 laser to desorb submolecular films of anthracene and naphthalene from a graphite surface. The desorbed aromatics are then irradiated with a pulse of light from a KrF excimer laser to give rise to a large population of ions. A problem with this system is that the fluence of the CO.sub.2 laser is very high, and contact of the laser beam with the graphite substrate gives rise to generation of C.sup.+ and C.sub.2.sup.+ ions directly from the graphite surface, even though the surface is cooled to -73.degree. C. ostensibly to prevent this.
The Becker et al. work is directed to a process referred to as surface analysis by laser ionization or "SALI". In the process an ion or laser beam sputters or desorbs material such as elemental metals or metal hydride from a surface (generally a metal surface). Next the neutral material released from the surface is irradiated with a focused high intensity burst of nonresonant multiphoton ionization energy. Then in a third step these ions are accelerated forward, focused, and allowed to drift in a field-free region for the time-of-flight detection. Again, this process has the failing that the desorption conditions are so harsh that they give rise to a background of secondary ions sputtered from the surface. In the second Becker et al. paper, resonant multiphoton ionization is compared with the nonresonant ionization which was shown in the first paper, with the results suggesting that nonresonant ionization is equal or superior to resonant multiphoton ionization. Becker et al. addresses elemental analyses and fails to demonstrate quantitation. The Nogar et al. system is also designed to address elemental detection. Tembreull et al. and Schlag and coworkers have developed methods which use laser desorption from a solid sample into a supersonic jet of a carrier gas for transport to a separate ionization zone. However, due to the increase of complexity in the passage of molecules from desorption region to ionization region, transmission efficiency of desorbed molecules significantly decreases and is not constant at a fixed experimental condition. Because of these problems, these systems do not permit quantitation.
Tembreull et al. describes an additional ion generation system which does not involve gas jet transport of desorbed molecules to the ionization zone. This system was only briefly reported. apparently being less preferred than the gas jet system. The system as described is similar to the present system in some respects but has the failing of employing a long period (200 .mu.sec) between desorption and ionization. Over this period, the cloud of desorbed molecules can disperse and cut down the number of ions generated. This system has other failings, as well. It uses a metal rod as its surface for presenting the test material. The beams of its desorption laser and ionizing laser are coaxial in the sample area. This geometry permits the ionizing laser beam to contact the solid surface carrying the sample and thus to generate additional particles which interfere with accuracy. In addition, this system uses desorption laser power levels which can fragment the molecules being examined and give rise to interferring ionic species.
While these background references represent a substantial body of progress in the field, they also reflect a need for further development. Thus further development is needed to provide a laser-based device which would generate volatilized molecules of nonvolatile materials and ionize the volatilized molecules with such reproducibility that quantitative analyses based on the ions so produced would be possible, especially at the femtomole level or lower. This represents much improved sensitivities. (For example, the SIMS method works at the 100 picomole level). It would further be desirable to have a method and apparatus which would generate quantitative amounts of ions over a substantial range and preferably with linearity over the substantial range. It would also be desirable to have a method and apparatus which would interface well with other analytical methodologies, e.g., liquid chromatography (LC) and would permit samples to be easily introduced into a laser-based ion generator so as allow analyses of the ions so formed to be carried out quickly and easily.
The present invention answers these needs by providing quantitation With high sensitivities, reproducibility and ease of sample handling in the laser-based ion generator.